Terence R.S. Ozolinš

Defects in embryonic development of EGLN1/PHD2 knockdown transgenic mice are associated with induction of Igfbp in the placenta.

The HIF (hypoxia inducible factor) hydroxylases EGNL1/PHD2 has been implicated in embryonic development. Here we knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. The partial inhibition of EGLN1 in utero is embryonic lethal in some, but not all mice on gestation day 14, and is associated with defects in placental and heart development, similar to those noted in the EGLN1 knockout mouse. Importantly, the in utero inhibition of EGNL1 varied greatly between the embryo proper and the placenta. Using this as a tool we show that the embryopathic effects are associated with knockdown of EGNL1 and the associated induction of Igfbp1 (insulin-like growth factor binding protein-1) mRNA in the placenta, but not the embryo.

Not a Walk in the Park: The ECVAM Whole Embryo Culture Model Challenged With Pharmaceuticals and Attempted Improvements With Random Forest Design

BACKGROUND The European Committee for the Validation of Alternative Methods (ECVAM) supported the development of a linear discriminant embryotoxicity prediction model founded on rat whole embryo culture (Piersma et al. (2004). Altern Lab Anim 32:275–307). Our goals were to (1) assess the accuracy of this model with pharmaceuticals, and (2) to use the data to develop a more accurate prediction model. METHODS Sixty-one chemicals of known in vivo activity were tested. They were part of the ECVAM validation set (N513), commercially available pharmaceuticals (N531), and Pfizer chemicals that did not reach the market, but for which developmental toxicity data were available (N517). They were tested according to the ECVAM procedures. Fifty-seven of these chemicals were used for Random Forest modeling to develop an alternate model with the goal of using surrogate endpoints for simplified assessments and to improve the predictivity of the model. RESULTS Using part of the ECVAM chemical test set, the ECVAM prediction model was 77% accurate. This approximated what was reported in the validation study (80%; Piersma et al. (2004). Altern Lab Anim 32:275–307). However, when confronted with novel chemicals, the accuracy of the linear discriminant model dropped to 56%. In an attempt to improve this performance, we used a Random Forest model that provided rankings and confidence estimates. Although the model used simpler endpoints, its performance was no better than the ECVAM linear discriminant model. CONCLUSIONS This study confirms previous concerns about the applicability of the ECVAM prediction model to a more diverse chemical set, and underscores the challenges associated with developing embryotoxicity prediction models.

Noninvasive high-resolution ultrasound reveals structural and functional deficits in dimethadione-exposed fetal rat hearts in utero.

BACKGROUND We previously showed dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, induces ventricular septation defects (VSD) and other heart anomalies in rat (Weston et al., 2011). Because of the relationship between cardiac structure and function, we hypothesized that DMO-induced structural defects of the heart are associated with in utero functional deficits. To test the hypothesis, the goals were (1) define the parameters for ultrasound in the rat conceptus, and; (2) use ultrasound to identify structural and functional deficits following DMO treatment. METHODS Different ultrasound modes (B-mode, M-mode, and Pulse-wave Doppler) using four high-resolution ultrasound transducer heads of varying frequency (25-40 MHz) were tested on gestational day (GD) 14, 15, 16, 17, and 21. Having identified the optimal conditions, pregnant Sprague-Dawley rats were administered six 300 mg/kg doses of DMO every 12 hr beginning at 19:00 hr on GD 8 to generate conceptuses with a high incidence of VSD. RESULTS The three ultrasound modalities were used to identify VSD and several novel and rare structural heart anomalies (cardiac effusions and bifurcated septum) in live rat fetuses. DMO-treated hearts had an array of functional deficits including a decrease in mean heart rate, ejection fraction, and cardiac output and increased incidence of bradycardia and dysrhythmia. CONCLUSIONS The ultrasound biomicroscope is an effective tool for the real-time characterization of the structure and function of embryo/fetal rat hearts. DMO causes significant deficits to in utero heart function for up to ten days (GD 21) following its final administration, suggesting long-term or possible permanent changes cardiac function.

Co-variation in frequency and severity of cardiovascular and skeletal defects in Sprague-Dawley rats after maternal administration of dimethadione, the N-demethylated metabolite of trimethadione

BACKGROUND The anticonvulsant trimethadione is a potent inducer of ventricular septation defects, both clinically and in rodents. Teratogenicity requires its N-demethylation to dimethadione, the proximate teratogen. It was previously demonstrated trimethadione only induced membranous ventricular septation defects in rat (Fleeman et al., 2004), and our present goal is to determine whether direct administration of dimethadione increases the incidence and severity of septation defects. METHODS Pregnant Sprague-Dawley rats were divided into five groups and administered either distilled water (control) or four different regimens of dimethadione. The core treatment was 300 mg/kg dimethadione b.i.d. on gestation day 9, 10 with additional groups given one additional dose of dimethadione 12 hr earlier, 12 hr later or two additional doses 12 hr earlier and later. Caesarian sections occurred on gestation day 21 and fetuses were examined for standard developmental toxicity endpoints. RESULTS The broadest dosing regimen yielded the highest incidence and the most severe heart and axioskeletal findings with a decrease in mean fetal body weight. The overall incidence of ventricular septation defects was 74%, of which 68% were membranous and 9% muscular. Outflow tract anomalies (17%) were also observed, as were malformations of the axioskeleton (97%), but not of the long bones, and of particular interest was the high incidence of sternoschesis. CONCLUSIONS Unlike trimethadione, dimethadione induces more serious muscular septation defects that are believed to be more clinically relevant. This, when taken together with the high incidence of total septation anomalies suggests dimethadione is useful for the study of chemically induced ventricular septation defects.

Moderate Exercise Attenuates Lipopolysaccharide-Induced Inflammation and Associated Maternal and Fetal Morbidities in Pregnant Rats.

Fetal growth restriction (FGR) and coagulopathies are often associated with aberrant maternal inflammation. Moderate-intensity exercise during pregnancy has been shown to increase utero-placental blood flow and to enhance fetal nutrition as well as fetal and placental growth. Furthermore, exercise is known to reduce inflammation. To evaluate the effect of moderate-intensity exercise on inflammation associated with the development of maternal coagulopathies and FGR, Wistar rats were subjected to an exercise regime before and during pregnancy. To model inflammation-induced FGR, pregnant rats were administered daily intraperitoneal injections of E. coli lipopolysaccharide (LPS) on gestational days (GD) 13.5–16.5 and sacrificed at GD 17.5. Control rats were injected with saline. Maternal hemostasis was assessed by thromboelastography. Moderate-intensity exercise prevented LPS-mediated increases in white blood cell counts measured on GD 17.5 and improved maternal hemostasis profiles. Importantly, our data reveal that exercise prevented LPS-induced FGR. Moderate-intensity exercise initiated before and maintained during pregnancy may decrease the severity of maternal and perinatal complications associated with abnormal maternal inflammation.

In Utero Dimethadione Exposure Causes Postnatal Disruption in Cardiac Structure and Function in the Rat

In utero exposure of rat embryos to dimethadione (DMO), the N-demethylated teratogenic metabolite of the anticonvulsant trimethadione, induces a high incidence of cardiac heart defects including ventricular septal defects (VSDs). The same exposure regimen also leads to in utero cardiac functional deficits, including bradycardia, dysrhythmia, and a reduction in cardiac output (CO) and ejection fraction that persist until parturition (10 days after the final dose). Despite a high rate of spontaneous postnatal VSD closure, we hypothesize that functional sequelae will persist into adulthood. Pregnant Sprague Dawley rats were administered six 300 mg/kg doses of DMO, one every 12 h in mid-pregnancy beginning on the evening of gestation day 8. Postnatal cardiac function was assessed in control (CTL) and DMO-exposed offspring using radiotelemetry and ultrasound at 3 and 11 months of age, respectively. Adult rats exposed to DMO in utero had an increased incidence of arrhythmia, elevated blood pressure and CO, greater left ventricular volume and elevated locomotor activity versus CTL. The mean arterial pressure of DMO-exposed rats was more sensitive to changes in dietary salt load compared with CTL. Importantly, most treated rats had functional deficits in the absence of a persistent structural defect. It was concluded that in utero DMO exposure causes cardiovascular deficits that persist into postnatal life in the rat, despite absence of visible structural anomalies. We speculate this is not unique to DMO, suggesting possible health implications for infants with unrecognized gestational chemical exposures.

Maternal rat serum concentrations of dimethadione do not explain intra-litter differences in the incidence of dimethadione-induced birth defects, including novel findings in foetal lung

To investigate mechanisms of chemical-induced congenital heart defects (CHD) we have developed a rat model using dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant, trimethadione (TMD). Dosing pregnant rats with 300mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses) produces a mean 74% incidence of CHD with inter litter variability ranging from 40 to 100%. The goal of this study was to determine if the variability in maternal serum concentrations of DMO on GD 14, a surrogate marker for total exposure, was related to the inter-litter differences in teratogenic outcomes. To test this hypothesis, pregnant rats were dosed as described above and serum levels of DMO assessed on GD 14. On GD 21, foetuses were collected by caesarean section, assessed for a number endpoints and the outcomes were correlated with the GD 14 serum concentrations of DMO. DMO exposure was associated with decreased foetal body weight, increased incidence of sternal defects and CHD, but these endpoints were not meaningfully correlated with maternal concentrations of DMO. Novel findings were decreased viability as measured one-hour following caesarean section, and delayed alveolar maturation. The major conclusions from these studies were first, that serum DMO concentrations on GD 14 did not predict teratogenicity, and second, delayed lung development may contribute to the decreased survival of foetuses at the time of caesarean section.

Analysis of the role of the HIF hydroxylase family members in erythropoiesis

HIF (hypoxia-inducible factor) hydroxylases have been implicated in EPO (erythropoietin) production and erythropoiesis. Here we examined the role of each of the three EGLN family members and the HIF asparaginyl hydroxylase FIH (factor inhibiting HIF) in EPO production. We examined the effect of inhibiting individual as well as combinations of HIF hydroxylases with RNAi. We found that inhibition of EGLN1 (egl nine homolog 1) as well as other members of the EGLN family (EGLN2 and EGLN3) led to accumulative EPO production in vitro. We then knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. Among all the in vitro and in vivo studies, no or minimal VEGF (vascular endothelial growth factor) mRNA or protein stimulation resulted from inhibition of EGLN1.

Cysteine-independent activation/inhibition of heme oxygenase-2.

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

In Utero Exposure to a Cardiac Teratogen Causes Reversible Deficits in Postnatal Cardiovascular Function, But Altered Adaptation to the Burden of Pregnancy

Congenital heart defects (CHD) are the most common birth anomaly and while many resolve spontaneously by 1 year of age, the lifelong burden on survivors is poorly understood. Using a rat model of chemically induced CHD that resolve postnatally, we sought to characterize the postnatal changes in cardiac function, and to investigate whether resolved CHD affects the ability to adapt to the increased the cardiovascular (CV) burden of pregnancy. To generate rats with resolved CHD, pregnant rats were administered distilled water or dimethadione (DMO) [300 mg/kg b.i.d. on gestation day (gd) 9 and 10] and pups delivered naturally. To characterize structural and functional changes in the heart, treated and control offspring were scanned by echocardiography on postnatal day 4, 21, and 10-12 weeks. Radiotelemeters were implanted for continuous monitoring of hemodynamics. Females were mated and scanned by echocardiography on gd12 and gd18 during pregnancy. On gd18, maternal hearts were collected for structural and molecular assessment. Postnatal echocardiography revealed numerous structural and functional differences in treated offspring compared with control; however, these resolved by 10-12 weeks of age. The CV demand of pregnancy revealed differences between treated and control offspring with respect to mean arterial pressure, CV function, cardiac strain, and left ventricular gene expression. In utero exposure to DMO also affected the subsequent generation. Gd18 fetal and placental weights were increased in treated F2 offspring. This study demonstrates that in utero chemical exposure may permanently alter the capacity of the postnatal heart to adapt to pregnancy and this may have transgenerational effects.